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Production of glycoprotein vaccines in Escherichia coli.

Ihssen J, Kowarik M, Dilettoso S, Tanner C, Wacker M, Thöny-Meyer L - Microb. Cell Fact. (2010)

Bottom Line: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens.It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction.The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

View Article: PubMed Central - HTML - PubMed

Affiliation: Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Gallen, Switzerland.

ABSTRACT

Background: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.

Results: Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.

Conclusions: The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

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Fed-batch cultivation of AcrA-O1 producing E. coli with semi-defined glycerol medium using three different feed and induction strategies. Strategy A - filled symbols: Linear feed of glycerol and tryptone from time -3 to 0 h, pulse of IPTG (1 mM) and L-arabinose (2 g L-1) at time 0 h, linear feed of glycerol, tryptone, L-rabinose (2.75 g L-1 h-1) and IPTG (80 μM h-1) from time 0 to 15 h. Strategy B - open symbols: Pulse of glycerol, tryptone, L-arabinose (4 g L-1) and IPTG (1 mM) at time 0 h, pulse of glycerol, tryptone and L-arabinose (4 g L-1) at time 4 h. Strategy C - shaded symbols: Pulse of glycerol, yeast extract and tryptone at time -2.6 h; pulse of glycerol, tryptone, L-arabinose (10 g L-1) and IPTG (1 mM) at time 0 h; linear feed of tryptone, L-arabinose (1.6 g L-1 h-1) and IPTG (10 μM h-1) from time 0 to 15 h. (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares), time 0 h (broken line): induction with L-arabinose and IPTG. (B) Time course of AcrA and AcrA-O1 formation. Normalized total cell protein samples were analysed by Western blot with anti-AcrA antibodies, numbers indicate time after induction, SF: samples from LB shake flask cultures. Lane B: same sample of strategy B as in middle blot, analysed on a blot with samples of strategy C (shorter development time, non-relevant lanes removed). E. coli CLM24 (pMIK44, pGVXN64, pGVXN114).
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Figure 5: Fed-batch cultivation of AcrA-O1 producing E. coli with semi-defined glycerol medium using three different feed and induction strategies. Strategy A - filled symbols: Linear feed of glycerol and tryptone from time -3 to 0 h, pulse of IPTG (1 mM) and L-arabinose (2 g L-1) at time 0 h, linear feed of glycerol, tryptone, L-rabinose (2.75 g L-1 h-1) and IPTG (80 μM h-1) from time 0 to 15 h. Strategy B - open symbols: Pulse of glycerol, tryptone, L-arabinose (4 g L-1) and IPTG (1 mM) at time 0 h, pulse of glycerol, tryptone and L-arabinose (4 g L-1) at time 4 h. Strategy C - shaded symbols: Pulse of glycerol, yeast extract and tryptone at time -2.6 h; pulse of glycerol, tryptone, L-arabinose (10 g L-1) and IPTG (1 mM) at time 0 h; linear feed of tryptone, L-arabinose (1.6 g L-1 h-1) and IPTG (10 μM h-1) from time 0 to 15 h. (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares), time 0 h (broken line): induction with L-arabinose and IPTG. (B) Time course of AcrA and AcrA-O1 formation. Normalized total cell protein samples were analysed by Western blot with anti-AcrA antibodies, numbers indicate time after induction, SF: samples from LB shake flask cultures. Lane B: same sample of strategy B as in middle blot, analysed on a blot with samples of strategy C (shorter development time, non-relevant lanes removed). E. coli CLM24 (pMIK44, pGVXN64, pGVXN114).

Mentions: Two fed-batch strategies were evaluated first: linear nutrient feed (strategy A) and two consecutive nutrient and inducer pulses (strategy B). With both strategies, 30-fold higher final optical densities (600 nm) compared to LB shake flask cultures were reached for the AcrA-O1 producing strain (Table 1, Figure 5A). However, strategy A with induction at an OD600 of 47 failed to yield sufficient amounts of glycosylated protein (Figure 5B). In contrast, Strategy B with induction at an OD600 of 14 yielded AcrA-O1 in comparable amounts as in LB shake flask (Figure 5B). A possible factor which influenced glycosylation was the specific growth rate at induction, which was 2-fold lower with strategy A compared to strategy B (Table 1, see also logarithmic growth curves in Figure 5A). Strategy C with induction at an intermediate cell density (OD600 = 30) and addition of two nutrient pulses followed by a linear feed of L-arabinose and tryptone lead to the highest levels of glycosylation in fed-batch culture (Figure 5B). In contrast to strategy A, antibiotics were included in the linear feed of strategy C which might have improved plasmid retention, and thus glycoconjugate formation.


Production of glycoprotein vaccines in Escherichia coli.

Ihssen J, Kowarik M, Dilettoso S, Tanner C, Wacker M, Thöny-Meyer L - Microb. Cell Fact. (2010)

Fed-batch cultivation of AcrA-O1 producing E. coli with semi-defined glycerol medium using three different feed and induction strategies. Strategy A - filled symbols: Linear feed of glycerol and tryptone from time -3 to 0 h, pulse of IPTG (1 mM) and L-arabinose (2 g L-1) at time 0 h, linear feed of glycerol, tryptone, L-rabinose (2.75 g L-1 h-1) and IPTG (80 μM h-1) from time 0 to 15 h. Strategy B - open symbols: Pulse of glycerol, tryptone, L-arabinose (4 g L-1) and IPTG (1 mM) at time 0 h, pulse of glycerol, tryptone and L-arabinose (4 g L-1) at time 4 h. Strategy C - shaded symbols: Pulse of glycerol, yeast extract and tryptone at time -2.6 h; pulse of glycerol, tryptone, L-arabinose (10 g L-1) and IPTG (1 mM) at time 0 h; linear feed of tryptone, L-arabinose (1.6 g L-1 h-1) and IPTG (10 μM h-1) from time 0 to 15 h. (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares), time 0 h (broken line): induction with L-arabinose and IPTG. (B) Time course of AcrA and AcrA-O1 formation. Normalized total cell protein samples were analysed by Western blot with anti-AcrA antibodies, numbers indicate time after induction, SF: samples from LB shake flask cultures. Lane B: same sample of strategy B as in middle blot, analysed on a blot with samples of strategy C (shorter development time, non-relevant lanes removed). E. coli CLM24 (pMIK44, pGVXN64, pGVXN114).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2927510&req=5

Figure 5: Fed-batch cultivation of AcrA-O1 producing E. coli with semi-defined glycerol medium using three different feed and induction strategies. Strategy A - filled symbols: Linear feed of glycerol and tryptone from time -3 to 0 h, pulse of IPTG (1 mM) and L-arabinose (2 g L-1) at time 0 h, linear feed of glycerol, tryptone, L-rabinose (2.75 g L-1 h-1) and IPTG (80 μM h-1) from time 0 to 15 h. Strategy B - open symbols: Pulse of glycerol, tryptone, L-arabinose (4 g L-1) and IPTG (1 mM) at time 0 h, pulse of glycerol, tryptone and L-arabinose (4 g L-1) at time 4 h. Strategy C - shaded symbols: Pulse of glycerol, yeast extract and tryptone at time -2.6 h; pulse of glycerol, tryptone, L-arabinose (10 g L-1) and IPTG (1 mM) at time 0 h; linear feed of tryptone, L-arabinose (1.6 g L-1 h-1) and IPTG (10 μM h-1) from time 0 to 15 h. (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares), time 0 h (broken line): induction with L-arabinose and IPTG. (B) Time course of AcrA and AcrA-O1 formation. Normalized total cell protein samples were analysed by Western blot with anti-AcrA antibodies, numbers indicate time after induction, SF: samples from LB shake flask cultures. Lane B: same sample of strategy B as in middle blot, analysed on a blot with samples of strategy C (shorter development time, non-relevant lanes removed). E. coli CLM24 (pMIK44, pGVXN64, pGVXN114).
Mentions: Two fed-batch strategies were evaluated first: linear nutrient feed (strategy A) and two consecutive nutrient and inducer pulses (strategy B). With both strategies, 30-fold higher final optical densities (600 nm) compared to LB shake flask cultures were reached for the AcrA-O1 producing strain (Table 1, Figure 5A). However, strategy A with induction at an OD600 of 47 failed to yield sufficient amounts of glycosylated protein (Figure 5B). In contrast, Strategy B with induction at an OD600 of 14 yielded AcrA-O1 in comparable amounts as in LB shake flask (Figure 5B). A possible factor which influenced glycosylation was the specific growth rate at induction, which was 2-fold lower with strategy A compared to strategy B (Table 1, see also logarithmic growth curves in Figure 5A). Strategy C with induction at an intermediate cell density (OD600 = 30) and addition of two nutrient pulses followed by a linear feed of L-arabinose and tryptone lead to the highest levels of glycosylation in fed-batch culture (Figure 5B). In contrast to strategy A, antibiotics were included in the linear feed of strategy C which might have improved plasmid retention, and thus glycoconjugate formation.

Bottom Line: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens.It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction.The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

View Article: PubMed Central - HTML - PubMed

Affiliation: Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Gallen, Switzerland.

ABSTRACT

Background: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.

Results: Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.

Conclusions: The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

Show MeSH
Related in: MedlinePlus